Alexander S Paramonov

Publications (10)49.89 Total impact

• Source

  Available from: Marcel Blommers

  Dataset: supplemental materials

  Zakhar O Shenkarev, Ekaterina N Lyukmanova, Alexander S Paramonov, Lyudmila N Shingarova, Vladimir V Chupin, Mikhail P Kirpichnikov, Marcel J J Blommers, Alexander S Arseniev
• Article: Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

  Zakhar O Shenkarev, Ekaterina N Lyukmanova, Ivan O Butenko, Lada E Petrovskaya, Alexander S Paramonov, Mikhail A Shulepko, Oksana V Nekrasova, Mikhail P Kirpichnikov, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the SDS denatured state was achieved in the nanodiscs containing anionic saturated lipid DMPG and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid POPC. The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (< 20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.
  Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
• Article: Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers.

  Zakhar O Shenkarev, Sergey V Balandin, Kirill I Trunov, Alexander S Paramonov, Stanislav V Sukhanov, Leonid I Barsukov, Alexander S Arseniev, Tatiana V Ovchinnikova
  [show abstract] [hide abstract]
  ABSTRACT: The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.
  Biochemistry 06/2011; 50(28):6255-65. · 3.42 Impact Factor
• Source

  Available from: Marcel Blommers

  Article: Lipid-protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins.

  Zakhar O Shenkarev, Ekaterina N Lyukmanova, Alexander S Paramonov, Lyudmila N Shingarova, Vladimir V Chupin, Mikhail P Kirpichnikov, Marcel J J Blommers, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: The choice of a suitable detergent-based membrane mimetic is of crucial importance for high-resolution NMR studies of membrane proteins. The present report describes a new approach of detergent screening. It is based on the comparison of 2D (1)H,(15)N-correlation spectra of a protein in a membrane-bilayer "reference" medium and in "trial" detergent-based environments. The proposed "reference" medium is the lipid-protein nanodisc (LPN) representing nanoscale phospholipid bilayers wrapped around by apolipoprotein A-1. The set of zwitterionic (DPC, DMPC/DHPC), anionic (SDS, LMPG, LPPG), and weakly cationic (LDAO) detergent-based media was screened for their ability to represent the native structure of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP. The VSD/LPN complexes composed of saturated zwitterionic (DMPC), anionic (DMPG), or a mixture of unsaturated differently charged (POPC/DOPG, 3:1) lipids were used as reference. All assayed detergent media demonstrate similar CD spectra of the domain with a high level (approximately 60%) of overall helicity but different 2D NMR spectra. Using the reference spectrum of the VSD in LPN, we were able to choose the detergent composition in which the membrane-like structure of the VSD is preserved.
  Journal of the American Chemical Society 04/2010; 132(16):5628-9. · 9.91 Impact Factor
• Source

  Available from: Marcel Blommers

  Article: NMR structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel KvAP: implications for voltage gating.

  Zakhar O Shenkarev, Alexander S Paramonov, Ekaterina N Lyukmanova, Lyudmila N Shingarova, Sergei A Yakimov, Maxim A Dubinnyi, Vladimir V Chupin, Mikhail P Kirpichnikov, Marcel J J Blommers, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: The structure and dynamics of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP was studied by high-resolution NMR. The almost complete backbone resonance assignment and partial side-chain assignment of the (2)H,(13)C,(15)N-labeled VSD were obtained for the protein domain solubilized in DPC/LDAO (2:1) mixed micelles. Secondary and tertiary structures of the VSD were characterized using secondary chemical shifts and NOE contacts. These data indicate that the spatial structure of the VSD solubilized in micelles corresponds to the structure of the domain in an open state of the channel. NOE contacts and secondary chemical shifts of amide protons indicate the presence of tightly bound water molecule as well as hydrogen bond formation involving an interhelical salt bridge (Asp62-R133) that stabilizes the overall structure of the domain. The backbone dynamics of the VSD was studied using (15)N relaxation measurements. The loop regions S1-S2 and S2-S3 were found mobile, while the S3-S4 loop (voltage-sensor paddle) was found stable at the ps-ns time scale. The moieties of S1, S2, S3, and S4 helices sharing interhelical contacts (at the level of the Asp62-R133 salt bridge) were observed in conformational exchange on the micros-ms time scale. Similar exchange-induced broadening of characteristic resonances was observed for the VSD solubilized in the membrane of lipid-protein nanodiscs composed of DMPC, DMPG, and POPC/DOPG lipids. Apparently, the observed interhelical motions represent an inherent property of the VSD of the KvAP channel and can play an important role in the voltage gating.
  Journal of the American Chemical Society 04/2010; 132(16):5630-7. · 9.91 Impact Factor
• Article: Molecular insight into mechanism of antimicrobial action of the beta-hairpin peptide arenicin: specific oligomerization in detergent micelles.

  Tatiana V Ovchinnikova, Zakhar O Shenkarev, Sergey V Balandin, Kirill D Nadezhdin, Alexander S Paramonov, Vladimir N Kokryakov, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a beta-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a beta-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the beta-hairpin (CN upward arrow upward arrowNC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins.
  Biopolymers 06/2008; 89(5):455-64. · 2.87 Impact Factor
• Source

  Available from: Marcel Blommers

  Article: Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides.

  Ekaterina N Lyukmanova, Zakhar O Shenkarev, Alexander S Paramonov, Alexander G Sobol, Tatiana V Ovchinnikova, Vladimir V Chupin, Mikhail P Kirpichnikov, Marcel J J Blommers, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: In the present Communication we demonstrate the possibility to use high-resolution NMR for the investigation of membrane proteins in reconstituted high-density lipoprotein (rHDL) particles. The rHDL particles are nanoscale phospholipid bilayers wrapped around by a dimer of apolipoprotein A-1 (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Lett. 2002, 2, 853−856). In contrast to the commonly used spherical micelles, the rHDL particles incorporate a lipid bilayer like in biological membranes. These particles still undergo isotropic motion on the NMR time scale, providing the application of high-resolution NMR spectroscopy of the peptides and proteins embedded into their bilayer. As an example, the topology of the membrane-active peptide Antiamoebin-I in the bilayer of the rHDL particles was determined by using the lipid-soluble relaxation probe technique.
  Journal of the American Chemical Society 03/2008; 130(7):2140-1. · 9.91 Impact Factor
• Article: Antiamoebin I in Methanol Solution: Rapid Exchange between Right‐Handed and Left‐Handed 310‐Helical Conformations

  Zakhar O. Shenkarev, Alexander S. Paramonov, Kirill D. Nadezhdin, Eduard V. Bocharov, Irina A. Kudelina, Dmitry A. Skladnev, Andrey A. Tagaev, Zoya A. Yakimenko, Tatiana V. Ovchinnikova, Alexander S. Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe1-Aib-Aib-Aib-Iva-Gly-Leu-Aib8-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl16. By using the uniformly 13C,15N-labeled sample of Aam-I, the set of conformationally dependent J couplings and 3hJNC couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the 13Cβ nuclei (Δδ(13Cβ)) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe1–Gly6) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 310-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib8 residue. The 15N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib8 residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic ‘propensity’ possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.
  Chemistry & Biodiversity 05/2007; 4(6):1219 - 1242. · 1.80 Impact Factor
• Article: High stability of the hinge region in the membrane-active peptide helix of zervamicin: paramagnetic relaxation enhancement studies.

  Zakhar O Shenkarev, Alexander S Paramonov, Tamara A Balashova, Zoya A Yakimenko, Michael B Baru, Leila G Mustaeva, Jan Raap, Tatyana V Ovchinnikova, Alexander S Arseniev
  [show abstract] [hide abstract]
  ABSTRACT: Zervamicin IIB is a 16 amino acid peptaibol that forms voltage dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. Stability of the hinge region and intermolecular interactions were investigated in the N- and C-terminally spin-labelled peptide analogues. Intermolecular and intramolecular paramagnetic enhancement indicates that zervamicin behaves as a rigid helical rod in methanol solution. There are no high amplitude hinge-bending motions, and the peptaibol is monomeric up to concentration 1.5 mM. Stability of the hinge region illustrates the helix stabilising propensity of the Pro residue in membrane mimic environments and implies absence of significant conformational rearrangement due to voltage peptaibol activation.
  Biochemical and Biophysical Research Communications 12/2004; 325(3):1099-105. · 2.48 Impact Factor
• Article: 1-Phenylethynylpyrene (1-PEPy) as refined excimer forming alternative to pyrene: case of DNA major groove excimer.

  Irina V Astakhova, Andrei D Malakhov, Irina A Stepanova, Alexey V Ustinov, Stanislav L Bondarev, Alexander S Paramonov, Vladimir A Korshun
  [show abstract] [hide abstract]
  ABSTRACT: 1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.5 ns), it is able to form inter- and intrastrand excimers on DNA, probably resulting from spatial preorganization of two dye molecules.
  Bioconjugate Chemistry 18(6):1972-80. · 4.93 Impact Factor